CN102281900A

CN102281900A - Stabilising excipient for inactivated whole-virus vaccines

Info

Publication number: CN102281900A
Application number: CN2010800046791A
Authority: CN
Inventors: A.弗兰康; M.切瓦利耶; N.莫雷诺; E.卡尔沃萨; S.齐加里尼; V.法布雷
Original assignee: Sanofi Pasteur SA
Current assignee: Sanofi Pasteur SA
Priority date: 2009-10-07
Filing date: 2010-10-07
Publication date: 2011-12-14
Anticipated expiration: 2030-10-07
Also published as: RU2012118386A; AU2010304898B2; US20110081380A1; PL2485766T3; PE20121405A1; MX2012003935A; AU2010304898A1; BR112012008000B1; EP3915586A1; EP2485766B1; AR078558A1; RU2560675C2; BR112012008000A2; WO2011042663A1; MX338300B; US8557253B2; CN102281900B; US20140037684A1; PT2485766T; MA33723B1

Abstract

The invention relates to a vaccine composition containing: (a) inactivated whole virus; and (b) a stabilising excipient comprising (i) a buffer solution, (ii) a mixture of essential and non-essential amino acids, (iii) a disaccharide, (iv) a polyol, (v) a chelating agent, (vi) urea or a urea derivative, and (vii) a non-ionic surfactant.

Description

The stabilising carriers of the whole virus vaccine of deactivation

The present invention relates to a kind of vaccine combination, it contains this inactivated whole virus and makes the stable excipient of this vaccine combination, this vehicle composition contain buffer, essential and nonessential ispol, disaccharide, polyhydric alcohol, chelating agen, urea or urea derivative and nonionic surfactant.The invention still further relates to the preparation method and the vehicle composition like this of this vaccine combination.

In the scope of viral vaccine, viral vaccine is divided into 3 types usually: live-virus vaccine, inactivated whole virus vaccines and subunit viral vaccine.These 3 types of vaccines are inherent by them and they are distinguished for some features of the vehicle composition of its preservation regulation use.Generally speaking, these subunit vaccines that contain the limited amount virus antigen are than the easier preservation of the whole virus vaccine of deactivation, even also all attempt to keep its structural intergrity because they are killed, or than the easier preservation of these live-virus vaccines, because they also attempt to keep its viral infection ability.Prepared appropriate excipients for every viroid vaccine.For the viral vaccine of these attenuated virus, according to waiting that preserving the attenuated virus prepared product has prepared different stabilising carriers.JP 57007423 has described that a kind of to be used for making stable disaccharide and the polyhydric alcohol with at phosphate buffer of attenuated strain of Measles virus be the excipient of base.EP 065905 has described contain that in phosphate buffer one or more are selected from ten monoamino-acids, lactose and Sorbitol amino acid whose and has been used to the excipient that makes the dengue virus attenuated strain stable.At last, EP 0869814 describes a kind of vaccine combination, and it contains a kind of chickenpox virus attenuated strain and stabilizing agent, and this stabilizing agent contains Sorbitol, mannitol, sucrose, glucosan, amino acid whose mixture, urea and EDTA.

In the field of inactivated virus vaccine, the vaccine combination of instant administration often contains protein for animal, for example cattle or people's albumin, gelatin or casein.These albumen become known for improving these vaccines, especially contain the preservation (Stabilization) that is difficult to preserve viral vaccine.The vaccine combination that contains the deactivation rabies virus is this situation.Frazatti-Gallina and colleague thereof are in " Vaccine " (2004), and the 23rd volume is emphasized the important function of protein in stablizing antirabic vaccine, because this excipient contains albumin in the 511-517 page or leaf.

Yet, in these vaccines, there is albumen, if do not have strictness to be controlled and may have the potential danger that spreads disease the potential irritated risk that also can exist a kind of effort to avoid except dietary protein origin, especially contain serum albumin when these vaccines, for example when albumin or albumin derivant.

Therefore, need to seek a kind of its and form and do not contain proteic stabilising carriers, especially so that to contain the vaccine of the inactivated whole virus (for example rabies virus) that is difficult to preserve stable so that the viral vaccine of these inactivated whole virus is stable.

Also need to seek a kind of stabilising carriers of making the low dosage inactivated virus vaccine stable of also being suitable for, promptly it contains a spot of total protein in the effective dose vaccine.For the very pure vaccine that contains considerably less residual protein impurity, this requirement is prior.These residual protein impurity even they have potential anaphylaxis risk, but can help to make the low dosage inactivated virus vaccine stable to a certain extent.They can stop virus gathering, structure degradation or adsorption phenomena that vaccine potency is reduced to take place.

For this reason, the objective of the invention is a kind of vaccine combination, it contains:

A) inactivated whole virus prepared product, and

B) stabilising carriers, it contains:

The i buffer,

The mixture of the essential and non essential amino acid of ii,

The iii disaccharide,

The iv polyhydric alcohol,

The v chelating agen,

Vi urea or urea derivative, and

The vii nonionic surfactant.

Preferably, this inactivated whole virus is a rabies virus.

According to an aspect of the present invention, this vaccine combination is also without any serum albumin.

According to another aspect, this vaccine combination is also without any the foreign protein of animal origin, preferably without any the outer product-derived of animal origin.

According to another aspect, these virus proteins are to have at least 70% of total protein in this vaccine combination.

According to another aspect, the total protein concentration in this vaccine combination≤100 μ g/ml, preferably≤80 μ g/ml.

One special aspect in, the total protein concentration that in the vaccine combination of an effective dose, contains≤100 μ g.

In aspect another is special, the amount of the total protein that contains in the vaccine combination of an effective dose is 1-50 μ g.

In another aspect of the present invention, this stabilising carriers is without any albumen, or any albumen and any peptide, or preferably without any albumen, any peptide and any oligopeptide.

According to another aspect, what contain in this vaccine combination must contain arginine or arginine salt and glutamic acid or glutamate, Glu at least with the mixture of non essential amino acid.

According to a special aspect, what contain in the vaccine combination of an effective dose must be 0.5-2.5mg with the amount of non essential amino acid.

According to another aspect, the disaccharide that contains in excipient is a maltose.

According to another aspect, the polyhydric alcohol that contains in this vaccine combination is a Sorbitol.

According to a special aspect, the disaccharide that contains in the vaccine combination of an effective dose and the amount of polyhydric alcohol are 10-50mg.

In one aspect of the method, the intercalating agent that contains in this vaccine combination is EDTA or edta salt.

According to a special aspect, the amount of the intercalating agent that contains in the vaccine combination of an effective dose is 0.01-0.1mg.

According to another special aspect, the urea that contains in the vaccine combination of an effective dose or the amount of urea derivative are 0.3-1.5mg.

In one aspect of the method, the nonionic surfactant that contains in this vaccine combination is poloxamer (poloxamere).

Preferably, this poloxamer is a poloxamer 188.

According to a special aspect, the nonionic surfactant amount that contains in the vaccine combination of an effective dose is 0.001-0.5mg.

According to another aspect, the pH of this vaccine combination is 7.0-10.0, preferably 7.0-9.0.

In one aspect of the method, the buffer that contains in this vaccine combination is phosphate buffer, Tris buffer, HEPES buffer or their mixture.

One special aspect in, this buffer is that its molar concentration is the phosphate buffer of 10-100mM.

The invention still further relates to a kind of vaccine combination preparation method, said composition contains the totivirus prepared product of purification and deactivation, wherein:

A) prepared the totivirus prepared product by results by the supernatant of the cell culture of this viral infection,

B) this totivirus prepared product is carried out purification and deactivation, perhaps this totivirus prepared product is carried out purification and deactivation, and

C) the totivirus prepared product with this purification and deactivation is diluted in the stabilising carriers, and this stabilising carriers composition contains:

The i buffer,

The mixture of the essential and non essential amino acid of ii,

The iii disaccharide,

The iv polyhydric alcohol,

The v chelating agen,

Vi urea or urea derivative, and

The vii nonionic surfactant.

According to a kind of optimal way, when not adding the outer product-derived of animal origin, implement method of the present invention.

In a kind of preferred implementation of the inventive method, this virus is rabies virus.

In one aspect of the method, method of the present invention comprises, after the totivirus prepared product dilution of purification and deactivation, the vaccine combination that so obtains is distributed in step in the packing device, and the step of freeze drying of this vaccine combination randomly.

The invention still further relates to a kind of vaccine combination, it contains the freeze-dried formulation purification that preferred implementation of with good grounds the inventive method obtains and the totivirus prepared product of deactivation.

The invention still further relates to a kind of stabilising carriers that is used for the whole virus vaccine of deactivation, its composition contains:

The i buffer,

The mixture of the essential and non essential amino acid of ii,

The iii disaccharide,

The iv polyhydric alcohol,

The v chelating agen,

Vi urea or urea derivative, and

The vii nonionic surfactant.

Preferably, this stabilising carriers compositions is also without any albumen, or any albumen and any peptide, or preferably without any albumen, any peptide and any oligopeptide.

Particularly preferably, the compositions of this stabilising carriers is also without any the animal origin product.

Detailed description of the present invention

Vaccine combination of the present invention contains this inactivated whole virus (or inactivated whole virus prepared product) and stabilising carriers, and its excipient is formed mixture, disaccharide, polyhydric alcohol, chelating agen, urea or urea derivative and the nonionic surfactant that contains buffer, essential and non essential amino acid.This vehicle composition advantageously replaces containing the excipient of proteic prior art in it is formed.Especially valuably, it makes these vaccine combinations of being difficult to preserve stable, very especially, make that to contain this rabies virus stable as these vaccine combinations of inactivated whole virus, and it needn't add albumen.

Vaccine combination of the present invention all is stable under liquid dosage form, frozen liq dosage form or freeze-dried formulation, and this provides very large application flexibility.

It is biological activity in time of freezing, liquid or freeze-dried formulation that stabilising carriers of the present invention keeps this vaccine combination.Generally speaking, freezing vaccine combination storage temperature≤-35 ℃; Liquid dosage form compositions storage temperature is+2 ℃-+8 ℃, and freeze-dried formulation compositions storage temperature is+5 ℃ approximately.Advantageously, the vaccine combination of under unfavorable conditions, preserving, as liquid dosage form vaccine combination, or allow these vaccine combinations be subjected to a series ofly thawing and when freezing again, also remaining on viral organism activity and physical integrity in the vaccine combination+37 ℃ of preservations.

Form immunogenic amount (it is important for bringing out antiviral protection immunity) and/or estimate viral organism activity in this vaccine combination by measuring as time passes this virus by detecting its effectiveness in the sample testing in official recognition's animal model.Under the situation of the vaccine combination that contains the deactivation rabies virus, by measuring as time passes (or course of defrosting in) continuously virus titer (its be that basis estimate with the measurement result that is not degeneration form glycoprotein G concentration) and/or estimating the stability of this vaccine combination by the effectiveness that adopts the NIH official testing to detect this vaccine combination.In order to estimate glycoprotein G content, can adopt the ELISA method of sandwich-type, this method is discerned at least one, the comformational epitope of two glycoprotein G preferably, as described in Example 1.When the ELISA method of two comformational epitopes implementing identification glycoprotein G, usually the antibody that uses this rabies virus of neutralization is as coated antibody, this rabies virus identification is positioned at the comformational epitope (" Journal of Clinical investigation " (1989) of glycoprotein G antigen site II, the 84th volume, the 971-975 page or leaf), use the neutralizing antibody of discerning the comformational epitope that is positioned at Protein G antigen site III as disclosing antibody (" Biologicals " (2003), the 31st volume, the 9-16 page or leaf).These results represent with iu, because people adopt contrast NIBSC international with reference to carrying out gauged standard as reference usually.Also may adopt to be considered to the favourable alternate BIAcore technology of ELISA method, this technology is used for the surface plasma body resonant vibration of quantitative virus titer based on application.

Stabilising carriers of the present invention also works by the physical integrity that keeps virion.Its special virion that stops generates aggregation and/or its structural degradation as time passes.This can use the instrument (for example zetasizer Nano ZS (Malvern instrument)) that detects these aggregations to be controlled, and can be based upon the distribution curve of virion size in this vaccine combination.

This virus or viral prepared product are the totivirus particulate forms, and it generally adopts formalin, formaldehyde or the chemical treatment of β propanoic acid lactone to carry out deactivation.Also can adopt other ablation method of describing as in WO 2005/093049.Main employed inactivated whole virus prepared product contains envelope virus, because they often more are difficult to preserve, and needs to use the particular excipient compositions to guarantee its preservation.Relate to Japanese encephalitis's inactivated whole virus prepared product especially.Particularly preferably, these inactivated whole virus prepared products contain this rabies virus.

These viral prepared products are from generally being by the cutting of the form of extract of the supernatant of the cell culture of this viral infection.Can use the traditional culture medium that contains animal origin serum with the production cell stock solution, and infect these cells.Advantageously, these culture medium that are used for cell culture and infection do not contain serum albumin, do not have the animal origin product yet.In these culture medium the optional albumen that exists generally be the low-down low molecular weight protein (LMWP) of concentration (≤10KD) or more definitely say peptide, therefore this reduce irritated risk.For example with trade name VP SFM (InVitrogen), Opti Pro ^TMSerum-free (InVitrogen), Episerf (InVitrogen), Ex-cell MDCK (Sigma-Aldrich), Ex-Cell ^TMVero (SAFC biosciences), MP-BHK The culture medium that serum free (MP Biomedicals), SFC-10 BHK express serum free (Promo cell), SFC-20 BHK express protein free (Promo cell), HyQ PF Vero (Hyclone R é f. SH30352.02), Hyclone SFM4 Megavir sell, (Ham-F10, Ham-F12) Nutrient medium, leibovitz L-15 culture medium (Hyclone) are this situations for MDSS2 culture medium (Axcell biotechnology), modification DMEM Iscove culture medium (Hyclone), Ham.For example, these rabies virus cuttings are to be obtained by the Vero cell stock solution of having produced, use the viral infection culture medium to infect then, these culture medium are preferably without any serum albumin, any protein for animal, even without any the animal origin product, VP SFM culture medium for example.

Advantageously, the totivirus prepared product of the deactivation that contains in vaccine combination of the present invention is very pure.Normally, deactivation behind the first purification, perhaps first on the contrary deactivation then purification from the virus of some cuttings.Do not use serum albumin, do not using the foreign protein of animal origin, even do not use under the outer product-derived situation of animal origin, when implementing any production of this virus and purification step, vaccine combination advantageously of the present invention is without any serum albumin, so that farthest reduce the anaphylaxis risk, also without any the foreign protein of animal origin, even without any the animal origin product, so that farthest reduce the pathophoresis risk.About " albumen of animal origin or product ", should be appreciated that it is that its production method comprises that at least one wherein uses albumen or product from the step of animal or human's material.About " foreign protein or product ", should be appreciated that it is albumen or the product of in a step of virus production and/or purification process, introducing.For example, albumen that in this culture media composition, randomly contains or product, the enzyme that uses when virus production and/or purification step (as trypsin or benzonase) all is foreign protein or product.When its production method comprised that at least one uses step from animal or human's material, these foreign proteins or product were animal origins.When using some yeast, antibacterial or plant to be prepared, these foreign proteins or product are not animal origins when these foreign proteins or product adopt other method (for example use vegetable material, adopt chemosynthesis or adopt gene recombinaton).Albumen or product from cell (going out these viruses to obtain vaccine combination of the present invention by these cells produce) are endogenous protein or product on the contrary, because they and these viruses produce (or being salted out) simultaneously.

Within the scope of the invention, can advantageously implement to have the purification process of good especially performance, these methods cause and obtain very pure viral prepared product.Enumerate viral purification methods as an example, it comprises anion-exchange chromatography, then cation-exchange chromatography, the chromatography end of describing with WO 97/06243 of passing through the metal-chelating affinity again.The rabies virus that obtains by the supernatant of infection cell culture for purification and deactivation, it number is the method that 0952310 patent application is described that a kind of system of selection is to use what propose in France on April 14th, 2009, the super centrifugation step of step, saccharose gradient that it is included in cation-exchange chromatography step on the carrier that contains the polymethacrylates matrix of the isobutyl groups of grafting sulfo group thereon, handle with benzonase and the inactivation step of use β-propanoic acid lactone.The full rabies virus prepared product of resultant deactivation is pure especially, but more is difficult to preserve (stablizing), because its residual protein impurity is considerably less.Virus quantity is few in this vaccine production thing, and this difficulty is just bigger.

By this vehicle composition, vaccine combination of the present invention still is stable, even at total protein concentration≤100 μ g/ml ,≤80 μ g/ml, and≤50 μ g/ml, even≤also like this under the situation of 20 μ g/ml.Generally speaking, virus protein accounts at least 70% of the total protein that exists in vaccine combination of the present invention, and preferably at least 80% of total protein, particularly preferably at least 90% of total protein.In an effective vaccine dose, it has usually≤100 μ g or≤50 μ g, even≤total protein concentration of 20 μ g.As pointing out that residual DNA amount is every effective vaccine dosage＜100pg, preferably＜50pg.About " effective vaccine dosage (or vaccine combination of effective dose) ", should be appreciated that it is in the vaccination first time or strengthen the amount of bringing out the inactivation of viruses that contains in the necessary dosage of human or animal's protective immunity after the immunization method recommended in the vaccination scope and the immunization protocol administration.Under the situation of the vaccination of anti-rabies virus, by the official testing method of OMS approval, NIH method of testing (describing in the rabies monograph (WHO Technical Series Report 941-2007 January) of OMS) has been measured the effectiveness of antirabic vaccine.The antirabic vaccine dosage of deactivation is effective when it contains at least 2.5UI according to this test.According to these inoculations or the serum-vaccination regimen of common recommendation, bring out antilyssic protective immunity development by this dosage of people's intramuscular administration.

These " total proteins " are corresponding to all albumen that exists in this vaccine combination.They are the residual proteins that are not removed during with these virus proteins, purification, as the albumen of cell protein, cell culture and viral infection culture medium and the albumen that in this purification process, randomly adds (for example benzonase under the situation of the purification process of the above-mentioned rabies virus of picture) expression.Generally speaking, adopt the Bradford method that total protein is quantized.In order to quantize the ratio of virus protein in these total proteins, under degeneration and reductive condition, this vaccine combination sample is carried out electrophoresis on polyacrylamide gel, then with the announcement and the electrophoretogram photodensitometry of Coomassie blue.Under the situation of the vaccine combination that contains the full rabies virus of deactivation, the virus protein of representing with the RNA polymerase L of envelope glycoprotein G, nucleoprotein N, phosphoprotein P, stromatin M and dependenc RNA is easy to identify on polyacrylamide gel electrophoresis.The total protein concentration that contains in the effective dose of vaccine combination of the present invention generally is 1 μ g-50 μ g, preferably 2 μ g-20 μ g.Particularly preferably at least 70% of total protein, at least 75%, at least 80%, at least 85%, even at least 90% is virus protein.Excipient of the present invention is particularly advantageous, because it can make the vaccine combination that is difficult to preserve stable, for example contains the vaccine combination of the full rabies virus of deactivation, and finds in the effective vaccine agent in described compositions:

-account for the virus protein amount of total protein at least 70%, and/or

The total protein that-100 μ g are following, often the amount of total protein is 1 μ g-50 μ g, preferably 2 μ g-20 μ g.

Vehicle composition of the present invention is preferably without any albumen, any peptide, even any oligopeptide, and generally speaking do not contain the animal origin product with farthest reduce can be relevant with its application biology and/or irritated safety problem.

Within the scope of the invention, term " this stabilising carriers is without any albumen " should be appreciated that it is the stabilising carriers of a kind of its composition without any biomacromolecule, and this biomacromolecule contains the amino acid whose chain more than 50 that will be joined to one another by peptide bond.Term " this stabilising carriers is without any albumen and any peptide " should be appreciated that it is that it forms the stabilising carriers without any biomacromolecule, and this biomacromolecule contains the amino acid whose chain more than 20 that will be joined to one another by peptide bond.Term " this stabilising carriers is without any albumen, any peptide and any oligopeptide " should be appreciated that it is that it forms the stabilising carriers without any biomolecule, and this biomolecule contains by one or more peptide bonds the aminoacid that is joined to one another.For example contain two amino acid whose dipeptides that will be joined to one another and be not included in outside the stabilising carriers compositions without any albumen, any peptide and any oligopeptide, but can constitute a part without any the stabilising carriers compositions of albumen and any peptide by unique peptide bond.

The ispol that constitutes this vehicle composition part contains at least a essential amino acids (whole necessary aminoacid cystine in whole necessary aminoacid, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine are represented) and at least a non essential amino acid at whole non essential amino acid (whole non essential amino acid aspartic acids, glutamic acid, alanine, agedoite, glutamine, glycine, proline and serine are represented).These have sour functional aminoacid can be acid or salt form.Like this too for these basic aminoacid.

Preferably, excipient of the present invention contains arginine or arginine salt at least, for example hydrochlorate of arginine and glutamic acid, or glutamate, Glu, for example sodium glutamate.At last, this ispol preferably contains 1-12 kind essential amino acids, wherein has arginine or its salt at least, and 1-8 kind non essential amino acid wherein has glutamic acid or its salt at least.In this vaccine combination the aminoacid total concentration general≤20g/l, 2g/l-10g/l often.In the vaccine combination of the present invention of effective dose, the essential and non essential amino acid total amount≤5mg of this expression, preferably its amount is 0.5-2.5mg, particularly preferably 0.8-1.8mg.The amount of arginine (being hydrochloride form) generally is higher than 0.5 with the ratio of the total amino acid content that contains in excipient, and the ratio of the amount of glutamic acid (being the sodium glutamate form) and the total amino acid content that contains in excipient is less than 0.5.

Excipient of the present invention also contains disaccharide.Except the disaccharide that obtains by animal origin raw material (as breast).As the disaccharide that is suitable for the object of the invention, can enumerate sucrose, maltose or trehalose, but the maltose that preferably uses single maltose or share with other disaccharide (as sucrose).The polyhydric alcohol that also comprises in this vehicle composition is non-animal origin.Be mainly six alcohol, for example Sorbitol and/or mannitol.Preferably use Sorbitol, because mannitol has the crystalline defective of meeting when vaccine combination of the present invention is freeze-dried formulation.Disaccharide and polyhydric alcohol total concentration generally are 30g/l-100g/l in this vaccine combination.Preferably, excipient of the present invention contains maltose and Sorbitol.In the vaccine combination of an effective dose, this is usually corresponding to the amount of 10-50mg, preferably corresponding to the amount of 20-25mg.The amount of maltose generally is at least 70% of maltose and a Sorbitol total amount.

Intercalating agent also is one of component of excipient of the present invention.It is mainly EDTA (ethylenediaminetetraacetic acid) or its salt (Na ⁺, K ⁺Deng).EDTA or edta salt concentration generally are 0.02-0.5g/l, preferably 0.02-0.2g/l in this vaccine combination.In the vaccine combination of an effective dose, this is usually corresponding to 0.01-0.1mg, preferably 0.01-0.05mg, the particularly preferably amount of 0.01-0.04mg EDTA or edta salt.

Urea or urea derivative, for example allylurea, acetamide, methyl carbamate or butyl carbamate also constitute the part of this vehicle composition.Preferably, this vaccine combination contains the general 1g-10g/l of concentration, the preferably urea of concentration 1g-5g/l.In the vaccine combination of an effective dose, this is usually corresponding to 0.3-3mg, preferably 0.3-1.5mg, the particularly preferably amount of 0.4-1.2mg urea.

Excipient of the present invention also contains and helps the nonionic surfactant that makes this vaccine combination stable.Be not bound by theory, it helps to keep this viral biological activity, also by avoiding generating the physical integrity that the degraded of aggregation or virus structure helps to keep virion.Also can avoid this virus to be adsorbed on the container wall, especially true when these walls are made with glass or plastics especially.The nonionic surfactant that is suitable for the object of the invention is that the raw material with non-animal origin obtains, and aspect materia medica with by non-be compatible through the enteral administration product.As the nonionic surfactant class that is particularly suitable for the object of the invention, can enumerate poloxamer, they are oxirane and expoxy propane " block copolymer ", its chemical formula is HO (C ₂H ₄O) _a(C ₃H ₆O) _b(C ₂H ₄O) _aH, a representative ring oxirane unit number wherein, b representative ring Ethylene Oxide unit number.The molecular weight of these poloxamers generally is 1000-16000 dalton.Molecular weight with poloxamer of special benefit is 5000-15500 dalton.These poloxamers are with trade name Pluronic in particular Sell, wherein pluronic F68, promptly poloxamer 188, and its expression at room temperature is the poloxamer of solid-state form, about 1750 dalton of the molecular weight of its polyoxypropylene, and its polyoxyethylene partly accounts for about 80% of this molecule gross weight.In these poloxamers, special recommendation poloxamer 188 (pluronic F68).Randomly, can use sorbitan ester, especially with trade name Tween The sorbitan polyoxyethylene ester of selling is as Tween 20 (sorbitan polyoxyethylene (20) monolaurates, promptly polysorbate 20), or Tween 80 (sorbitan polyoxyethylene (20) monoleates, promptly polysorbate 80).The low-down concentration of nonionic surfactant that is fit to the object of the invention is used, and keeps the inactivation of viruses morphology of particles like this, its should be still close with size and live virus.Very during high concentration, this surfactant can dissociate or change the structure of these virion, and the structure of enveloped virus especially can influence the immunogenicity of this vaccine combination like this.Work as pluronic When F68 was used as surfactant, the total concentration≤1g/l in this vaccine combination generally was 0.005g/l-1g/l, particularly preferably 0.01g/l-0.1g/l.In this concentration range, 188 pairs of these immune systems of poloxamer are not brought into play assosting effect.In the vaccine combination of an effective dose, this is usually corresponding to 0.001mg-0.5mg, preferably the amount of 0.003-0.3mg.Most preferably, this vaccine combination contains poloxamer 188 or the mixture of poloxamer 188 and polysorbate 20 randomly.

Select excipient buffer of the present invention to make that the pH of this vaccine combination is 7.0-10.0, preferably 7.0-9.0, particularly preferably 7.3-8.3.Just in this pH scope, the physical heat stability of inactivation of viruses particle, particularly the physical heat stability of rabies virus particle is maximum.These studies show that to phasor when pH was 7.3-8.3, the temperature that this rabies virus prepared product must be heated at least 60 ℃ was just observed its rabies virus particle accumulation, and o'clock at room temperature observe serious clustering phenomena in pH＜6.0.Sort buffer liquid is phosphate buffer, Tris buffer, HEPES buffer or their mixture normally.Their concentration generally is 10-100mM.Preferably, this vehicle composition contains phosphate buffer, and its molar concentration is 10-100mM normally, or the phosphate buffer of form of mixtures and Tris buffer.

The particularly preferred vaccine combination of the present invention contains:

The full rabies virus prepared product of deactivation, and stabilising carriers, it contains:

Phosphate buffer;

The mixture of essential and non essential amino acid, it contains arginine or arginine salt and glutamic acid or glutamate, Glu at least;

Maltose;

Sorbitol;

EDTA or edta salt;

Urea;

Poloxamer 188;

The pH of this vaccine combination is 7.3-8.3.

Randomly, can be added to Tween 20 in this vaccine combination as additional nonionic surfactant.This vaccine combination advantageously also generally contains very pure rabies virus prepared product without any the outer product-derived of serum albumin and any animal origin.Concentration＜the 1g/l of poloxamer 188, most preferably 0.01g/l-0.1g/l in this vaccine combination.The molar concentration of phosphate buffer generally is 10-100mM.Arginine and glutamic acid or they salt separately is the most components in this ispol, because they are at least 2/3 of essential and non essential amino acid total amounts according to the weight meter generally.The concentration of the mixture of this essential and non essential amino acid is 2-10g/l normally, and the total concentration of maltose and Sorbitol is 50-100g/l normally, and the concentration of EDTA or edta salt is 0.02-0.2g/l normally, and the total concentration of urea is 1-5g/l normally.Total protein concentration 5-50 μ g/ml normally in this vaccine combination.In the antirabic vaccine of effective dose, its ordinary representation must be 0.5mg-2.5mg with the amount of non essential amino acid, the amount of maltose and Sorbitol is 10-50mg, EDTA or edta salt amount are 0.01mg-0.1mg, the amount of urea is 0.3-1.5mg, the amount of poloxamer 188 is 0.001-0.5mg, and the amount of total protein is 2-20 μ g.

The preparation method of vaccine combination of the present invention, except that viral prepared product production, purification and these steps of deactivation, also comprise the step that adds stabilising carriers in the totivirus prepared product of this purification and deactivation, and the compositions that so obtains is distributed in step in the packing device.Generally speaking, before being assigned to this vaccine combination in these packing devices, this prepared product is diluted in the stabilising carriers of the present invention.

Vaccine combination of the present invention can exist with dosage form in bulk (sous la forme de vrac): in this case, in case distribute, the concentration height that this inactivated whole virus concentration ratio exists in this vaccine combination, but this vehicle composition is same compositions.This dosage form in bulk generally remains on＜and freezing under-35 ℃ the temperature.At this moment by this dosage form in bulk is thawed, then it is diluted in this stabilising carriers to reach the virus concentration of expectation, carry out the allocation step of this vaccine combination.At this moment dosage form product in bulk to the end, it is redistributed in the packing device.Aseptic in order to ensure this vaccine combination, can also before allocation step, add a sterilising filtration step, for example use the sterilising filtration step of 0.2 μ m filter membrane.

Generally by obtaining this dosage form in bulk in purification and the diafiltration in buffer of inactivation of viruses prepared product implementing to obtain when purification process finishes, described buffer is generally corresponding to the buffer of this excipient.If for example the buffer of this excipient is the 50mM phosphate buffer, then use the 50mM phosphate buffer as diafiltration buffer.If expectation improves virus concentration, be ultrafiltration step before the diafiltration steps then.Use has the 5kDa-100kDa of being generally, 8kDa-50kDa preferably, and particularly preferably the low ultrafilter membrane of holding back threshold value of about 10kDa so that farthest this virus is retained in the trapped substance, and is removed the salt that negative effect can be arranged freeze drying process.Then, the trapped substance that contains viral prepared product is mixed with this stabilising carriers, to obtain dosage form in bulk (vrac), it is formed corresponding to vaccine of the present invention and forms.Can also use the buffer that has same composition with stabilising carriers as diafiltration buffer, in a step, to obtain dosage form in bulk.

The vaccine combination preparation method of the present invention that contains the full rabies virus of deactivation comprises the steps: to use the culture medium of cell culture and viral infection, and (this culture medium is preferably without any serum albumin and any animal origin product, for example as using VP SFM culture medium), by the supernatant of the Vero cell culture of viral infection, produce the totivirus prepared product by results.Being used on April 14th, 2009 especially is method purification and this virus of deactivation that 0952310 patent application is described in the patent No. of french application, this method be included in preferably contain based on the cation-exchange chromatography step on the carrier of the matrix of the polymethacrylates of the isobutyl groups of grafting sulfo group thereon, with reorganization benzonase handle, the inactivation step of the super centrifugation step of saccharose gradient and use β-propanoic acid lactone.The full rabies virus prepared product of the deactivation that obtains is very pure, without any the outer product-derived of serum albumin and any animal origin; Residual DNA content＜100pg/ml, these virus proteins are at least 70% of total proteins.Prepare dosage form in bulk according to the pattern of describing at leading portion then, it generally is kept at＜-35 ℃ temperature under.With the expectation virus concentration this dosage form in bulk is diluted in the excipient of the present invention again, then it is assigned in these packing devices.In each packing device, add this vaccine combination of 0.1ml-1ml usually, so that this compositions is in case distribution just contains at least one effective vaccine dosage.These are used to distribute the packing device of last dosage form product in bulk to be bottle (making with glass or plastics) or syringe form usually, but also can use and inoculate other compatible packing device of operation.

The grain size analysis (this Instrument measuring is based on the Brownian movement of the particle of light " accurate elasticity " scattering (dynamic light scattering)) of using z é tasizer Nano ZS instrument (Malvern Instrument) that the vaccine combination of the present invention that contains this purification rabies virus is carried out shows and exists single 100-300nm virion (the about 180nm of its meansigma methods) evenly group that it is corresponding to the average-size of wild rabies virus.In vaccine combination preservation process of the present invention, do not detect aggregation, do not detect the variation of virion size distribution curve yet.

The vaccine combination of these liquid dosage forms during at least 3 months, was stable (referring to embodiment 3) during at least 1 month under 23-27 ℃ under temperature 2-8 ℃.They also can preserve 12 months preferably at least 24 months at least during for freezing dosage form under≤-35 ℃ of temperature.When adopting traditional lyophilization, also can preserve these vaccine combinations with freeze-dried formulation.Vehicle composition of the present invention can not cause the significantly sacrificing of virus during step of freeze drying.A kind of freeze drying process is that said composition is freezing to being lower than under-40 ℃ the temperature, then with its product drying twice, dryly for the first time carries out under-15 ℃ of temperature approximately and 80 microbars, and dryly for the second time carries out under about+40 ℃ of temperature and 80 microbars.The residual moisture content of these freeze dried vaccine dosage≤3%, and at temperature 2-8 ℃ down stable at least 18 months (referring to embodiment 3).At last, as the antirabic vaccine compositions table of describing in the present invention reveal with trade name VeroraB ^TMThe same stability of the vaccine combination of the prior art of selling (its total protein concentration that contains in a dosage vaccine is to survey at least 100 times of total protein concentration (general≤50 μ g) in the vaccine combination of the present invention of an effective dose).In addition, vehicle composition of the present invention advantageously can be preserved the vaccine combination of liquid or freezing dosage form muchly, is freeze-dried formulation even preferably preserve dosage form.

This vaccine combination is a liquid dosage form when preserving, if or can directly deliver medicine to after thawing during its freezing preservation and treat vaccinated experimenter.When it is freeze-dried formulation, this lyophilized products is dissolved in the diluent that is generally saline solution immediately, for example as hypotonic sodium chloride solution.This diluent can also contain the very surfactant of low concentration, and its chemical constitution is compatible with non-the use through the intestinal approach aspect materia medica.It is Tween normally 20, Tween 80 or pluronic F68 uses them with the performance assosting effect with low-down concentration.Surfactant concentration in this diluent is (w/w) generally speaking≤0.1%.

When this vaccine is freeze-dried formulation, be generally the kit form of two packings, first (generally speaking being the doleiform formula) is equipped with this freeze dried vaccine compositions, and second (generally speaking being bottle or syringe form) is equipped with this diluent.Can also use " bypass " type syringe, wherein this vaccine combination is contained in the bottom of this syringe, and this diluent is adorned on its top.

The present invention relates to the whole virus vaccine that is used for deactivation at last, is used for the stabilising carriers of the full rabies virus vaccine of deactivation especially, and it contains:

A. buffer,

The mixture of b. essential and non essential amino acid,

C. disaccharide,

D. polyhydric alcohol,

E. chelating agen,

F. urea or urea derivative, and

G. nonionic surfactant.

Preferably, this excipient is without any albumen, preferably without any peptide and any albumen, also more preferably without any albumen, any peptide and any oligopeptide.Also most preferably without any the product of animal origin.

To understand the present invention better by reading following embodiment, these embodiment are used to the present invention is described and do not limit its content.

Fig. 1 be 3 in antirabic vaccine compositions (wherein this stabilising carriers compositions is a F04+0.001% poloxamer 188) thaw continuously with freeze cycle process again in adopt the overlapping of virion size distribution curve that " dynamic light scattering (DLS) " obtain.● D0 (before freezing), ■ D1 (in the back that thaws for the first time); ▲ D2 (back thaws for the second time) and+D3 (thaw for the third time back).

Fig. 2 be 3 in antirabic vaccine compositions (wherein this stabilising carriers compositions is a F04+0.01% poloxamer 188) thaw continuously with freeze cycle process again in adopt " dynamic light scattering (DLS) " to obtain virion size curves overlapped.● D0 (before freezing), ■ D1 (in the back that thaws for the first time); ▲ D2 (back thaws for the second time) and+D3 (thaw for the third time back).

Embodiment 1: excipient is to the influence of the dosage form vaccine combination stability in bulk of the rabies virus that contains purification and deactivation

1.1 the preparation of dosage form in bulk

By using the viral infection culture medium of serum-free, on the Vero cell, carried out rabies virus production.Make these Vero cell line cells be adapted to condition of culture as the serum-free medium of in WO 01/40443 application, describing.Then, they are transferred in the biomass generator, it is equipped with the cytodex 1 little carrier 1 (Invitrogen) in VP SFM culture medium.PH is about 7.2 ± 0.2 by keeping, oxygen saturation 25% ± 10% and to this culture medium carry out gentle agitation 37 ℃ cultivate 3-4 days down after, contain the viral infection culture medium of VP SFM culture medium (Invitrogen) by use, these cells have used rabies virus (Pitmann-Moore strain) to infect with infection multiplicity 0.01.At the 7th day (R1), the 11st day (R2) and the 15th day (R3) gathered in the crops the supernatant of infection cell culture.After each results, add its new viral infection culture medium again.Adopt twice continuous filtration to make the supernatant clarification of the culture that contains infective virus: to use prefilter (the Sartopure PP2 that makes by 8 μ m polypropylene for the first time, SARTORIUS), this prefilter is removed certain carrier, the Vero cell and the large scale cell debris that come off from carrier slightly that sucks in when results, (Sartopore 2 to use the PES filter of being made up of 0.8 μ m and two combination of filters of 0.45 μ m for the second time, SARTORIUS), it removes these aggregations.Based on the mensuration of the amount of the glycoprotein G (gpG) that adopts the ELISA method to carry out in the following manner, measured the amount of the rabies virus that in these clarification cuttings, exists:

In the hole of ELISA microwell plate, distribute about 0.12 μ g/100 μ l 1112-1 anti-gpG monoclonal anti liquid solution (at " Journal of Clinical investigation ", (1989), the 84th volume, this solution properties has been described) in the 971-975 page or leaf, this solution has been diluted in bag in advance and has been cushioned in the liquid (0.2M carbonate buffer, pH 9.6).Incubation then with lavation buffer solution (phosphate buffer that adds Tween20,0.05%) washing repeatedly, distributes the saturated buffer of 100 μ l (phosphate buffer that adds 1% bovine albumin serum) after one night in each hole in the cold house.After one hour, then washing has obtained a series of dilution factors of each sample to be tested in dilution buffer liquid (phosphate buffer that adds 0.05%Tween20 and 1% albumin serum) several times at 37 ℃ of incubations.Abreast, obtain a series of dilution factors of reference sample in each microwell plate, its contrast NIBSC international standard (for example PISRAV) is calibrated.At 37 ℃ of incubations after a hour again, then washing several times, (its characteristic is in " Biologicals " (2003) for the monoclonal antibody solution D 1 of distribution 100 μ l anti-gpG mices in each hole, the 31st volume, the 9-16 page or leaf obtains describing), it has carried out biotinylation and be diluted to 1/5000 back in dilution buffer liquid using.Allow these plates place 37 ℃ following 1 hour, in each hole, distribute then 100 μ l in advance in dilution buffer liquid dilution 1/5000 cyclic washing is several times before with the link coupled solution of streptavidin of peroxidase (Southern Biotechnology Associates).Incubation is after one hour again at 37 ℃, and then washing distributes 100 μ l 0.05M pH5 citrate buffers several times in each hole, and it contains announcement substrate (O-phenylenediamine).At incubation under the condition of lucifuge and room temperature after 30 minutes, by adding 50 μ l/ hole 2N H ₂SO ₄Solution is ended it and is disclosed reaction (r é action de r é v é lation).Locate to read the spectrophotometric number of microwell plate at two wavelength (492nm and 620nm).The optical density of measuring is two readings poor when considering that plastics absorb.Adopt the straight parallel collimation method to calculate relative activity according to the suggestion of European Pharmacopoeia (Pharmacop é e Europ é enne).This sample rabies virus titre is with the mensuration basis of the glycoprotein G concentration of this rabies virus, it with respect to it with reference to representing with UI/ml.

After also the electrical conductivity of clear liquor and pH being controlled, use 20mM Tris, 150mM NaCl, the equilibrated Fractogel of pH=7.5 buffer in advance EMD SO3 ^-Chromatograph carrier (Merck) is gone up the normal glycoprotein G of the deposition about 50UI of every ml carrier (adopting ELISA to measure).This chromatograph carrier washs with level pad subsequently, then, rabies virus is eluted in 20mM Tris, 600mM NaCl, the pH=7.5 buffer, reclaims the viral peak of independent fraction.Carrying out the ultrafiltration step on PES Medium Screen 100KD film (PALL) then, then is the diafiltration in 20mM Tris, NaCl 150mM, pH=7.5 buffer.Add MgCl again ₂Solution is so that the concentration in this percolate (diafiltrat) is 2mM.Add the rough cutting of 15U/ml by past this reaction culture medium, and allow this reaction culture medium under laboratory temperature, keep 2 hours, use the processing of benzonase.On 34-60% sucrose pad, use 45 type titanium rotors to carry out super centrifugal 2h down with 21000rpm at+5 ℃.Recovery contains the gradient fraction of purified virus, merges, and is diluted in then in 50mM phosphate, 150mM NaCl, the pH=7.5 buffer, so that last purified virus suspension vol is about 1/12.5 of a clarification cutting volume.Then, this purified virus suspension is handled by β propanoic acid lactone and is carried out deactivation, then makes β propanoic acid lactone deactivation 2 hours by heating under about 37 ℃ of temperature.By with 10kDa film (Omega medium screen PALL) ultrafiltration, then diafiltration in 50mM phosphate, 150mM NaCl, pH 7.5 buffer concentrates 6 times with the viral prepared product of purification and deactivation.The rabies virus prepared product that obtains is for concentrating dosage form in bulk, and its titre based on the mensuration of glycoprotein G concentration is about 80UI/ml.Come the first-class branch of dialysis dosage form in bulk in the face of one in 4 excipient (F01-F04) then, this excipient is formed as follows.

1.2 the composition of the excipient of test

F01:50mM phosphate, 150mM NaCl, maltose (5%, p/p), the pH=8.0 buffer;

F02:50mM phosphate, maltose (5%, p/p), the pH=8.0 buffer;

F03:50mM phosphate, maltose (5%, p/p), poloxamer 188 (BASF) (0.001%, p/p), the pH=8.0 buffer;

F04:489PM, pH=8.0 buffer, it is composed as follows.

。

By the contents melting in the bottle that 111.5g essential amino acids (Gibco, reference number 074-90680N, lot number 14773) is housed has been prepared essential amino acids solution at 5 liters with the acidifying water of 100ml 12N HCl (being used for the injectable prepared product).

By the contents melting in the bottle that 40.7g non essential amino acid (Gibco, reference number 074-90680N, lot number 14775) is housed has been prepared non essential amino acid solution at 5 premium on currency (being used for the injectable prepared product).

1.3 the test that 4 kinds of form formulas in bulk are carried out

This dosage form in bulk is formulated in 4 kinds of test vehicle compositions.By the titre (representing) of results of regular determination glycoprotein G with UI/ml estimated these four kinds prescriptions (vaccine combination) different storage temperatures-70 ℃ ,+5 ℃ and+37 ℃ under as time passes stability.What obtain the results are shown among Table I-III, and these results represent with UI/ml.

Table I: 4 kinds of form formulas in bulk are+37 ℃ stability

Time (week)	0	1	2
				F01*	41.25*	30.21	17.51
F02	60.41	23.15	5.12
				F03	60.29	23.16	5.2
F04	57.26	52.37	52.68

Table II: 4 kinds of form formulas in bulk are+5 ℃ stability

Time (week)	0	3	12	24
					F01	41.25**	39.71	45.56	38.55
F02	60.41	53.29	42.27	38.92
					F03	60.29	52.64	47.75	41.33
F04	57.26	62.19	61.06	61.32

Table III: 4 kinds of form formulas in bulk are-70 ℃ stability

Time (week)	0	3	12	24
					F01	41.25	33.81	37.61	27.59
F02	60.41	53.52	53.06	53.62
					F03	60.29	57.87	60.15	56.46
F04	57.26	57.48	59.92	56.56

*: F01, F02, F03 and F04 refer to vehicle composition, have prepared dosage form in bulk therein

*: represent with UI/ml.

These results show that excipient F04 is the stable excipient of rabies virus prepared product that can make purification and deactivation, and is especially true when storage temperature improves especially.This shows that also the excipient that contains sucrose (maltose) and nonionic surfactant (for example poloxamer 188) in pH 8.0 buffer is not enough to make effectively the rabies virus prepared product stable.

Embodiment 2: the effect of surfactant in stablizing the vaccine combination that contains the purification rabies virus

2.1 The preparation of vaccine combination

Adopt the same quadrat method of using with embodiment 1 to obtain the suspension of purification rabies virus.After super centrifugation step, this purified virus prepared product is very dense form.By adopting the Bradford method, its total protein concentration is 7mg/ml.This viral prepared product is distributed in 3 containers made from polypropylene, be diluted in then and attempt to estimate in 3 kinds of different excipient of its Stabilization, so that the last total protein concentration in each excipient of testing is reduced to 10 μ g/ml (dilution gfactor 1/700).The composition difference of 3 kinds of excipient of being tested only is their poloxamer 188 content, but all contains 489PM, pH=8.0 buffer, and it forms description in embodiment 1.The excipient of no poloxamer 188 has the same composition of the excipient F04 that describes with embodiment 1.Two other excipient has replenished 0.01g/l poloxamer 188 (called after excipient F04+0.01% poloxamer) and 0.1g/l poloxamer 188 (called after excipient F04+0.1% poloxamer) respectively.

2.2 Stability study and result

By they being placed under the disadvantageous preservation condition, promptly allow them be subjected to estimating the stability of 3 kinds of vaccine combinations according to a series of freezing of following proposal and thawing:

At (J0) on the same day that 3 kinds of vaccine combinations are freezing to-70 ℃.Then, each of 3 kinds of compositionss carries out thawing for 3 times under+5 ℃, then under-70 ℃ freezing again 7-14 hour, thaws at the 1st, 2 and 3 day (J1, J2 and J3 respectively) then.The stability of the vaccine combination after at every turn thawing is estimated by measuring the total virus protein concentration.Employing is carried out this mensuration based on the BIAcore technology of application surface plasma resonance.Also, some particle size distribution curves in continuous course of defrosting are analyzed by adopting light quasi-elastic scattering technology (dynamic light scattering or DLS technology).

For the coupling reagent kit of implementing the BIAcore technology, at first use to be provided by manufacturer (with reference to the amine coupling reagent kit, BR-1000-50), gets up monoclonal antibody D1 and the sensor chip coupling of anti-gpG Mus by covalent bond.Then, 30 μ l sample to be analyzed was expelled in this instrument automatically in the clock time at 3 minutes.The rabies virus that exists in this sample causes that with the monoclonal antibody interaction that is fixed on the sensor chip refractive index of this sensor chip surface changes, and it shows as the variation (RU) of tracer signal.These results that obtain carry out record with arbitrary unit (Δ RU), and compare with the result who uses a series of reference standards to obtain, and this reference standard contains the purification rabies virus of known quantity.The Δ RU that each sample is measured is based on being converted to the total virus protein concentration with the resulting standard curve of this reference.For each test sample, these mensuration are carried out (in quadruplicate) 4 times.After each the mensuration, by carrying out injection 10 μ l Glycine (10mM, pH=1.5) buffer solution for cleaning sensor chips continuously twice.The test vaccine compositions is resulting the results are shown in the Table IV to each.These values are represented with μ g/ml virus protein.

Table IV: thawing continuously for 3 times and the stability study of 3 kinds of vaccine combinations in the refrigerated periodic process again

*:, represent with μ g/ml based on the resulting virus protein average titer of mensuration that 4 times test sample is carried out

*: in bracket, represent based on the root variance that the mensuration of 4 times test sample being carried out is calculated.

These results clearly illustrate that the excipient F04 of no poloxamer can not effectively preserve the rabies virus prepared product, because measure the virus titer loss 88% between the titre behind D0 titre and thaw for the third time (D3).On the contrary, excipient F04 is in case unusual small amount of supplemental poloxamer 188, and the virus titer loss just reduces (when F04 replenished 0.001g/l poloxamer 188, the titre loss was 33%, and when F04 replenished 0.01g/l poloxamer 188, the titre loss was 23%) greatly.These results show that this excipient should contain component and the poloxamer 188 that exists simultaneously in the 489PM buffer, so that effectively preserve the vaccine combination that contains this rabies virus.

By use Zetasizer Nano Zs instrument (Malvern Instrument) to adopt the DLS method also to analyze every kind of particle size distribution curve after thawing in 3 kinds of vaccine combinations at every turn.In the disposable groove made from polystyrene, add 450 μ l sample to be analyzed, allow it accept one-wavelength laser radiation (HeNe laser instrument, λ=632.8nm) then.The signal of this instrument record is corresponding to the fluctuation of the scattered light that is caused by these particle Brownian movements.The data of these records have adopted computer software to handle.At last, these results represent with the form for the particle size distribution curve of each sample of being tested.2 figure represent the overlapping of 4 particle size distribution curves, they are to use respectively by the stable rabies virus compositions (Fig. 1) of excipient F04+0.001% poloxamer 188, and use by excipient F04+0.01% poloxamer 188 stable rabies virus compositions (Fig. 2) at J0 days (before freezing), J1, J2 with obtained in J3 days.

These two figure clearly illustrate that:

1) these particle size distribution curves are unimodal, and are Gauss distribution, the about 180nm of its intermediate value.This means that the virion subgroup in using this stable vaccine combination of excipient F04+0.001% poloxamer 188 or excipient F04+0.01% poloxamer 188 is uniform, do not contain aggregation, and contain and the similarly full rabies virus group of wild rabies virus group, and

2) be subjected to repeatedly thawing continuously and again during freezing processing, these particle size distribution curves do not change when these rabies virus vaccine combinations.

In this vehicle composition, exist nonionic surfactant (as poloxamer 188) also can keep the physical integrity of rabies virus group, even place abominable preservation condition following time also like this when this vaccine combination.

2.3 Research to the freeze dried vaccine composition stable

2.3.1: the freeze dried vaccine preparation of compositions

At first use the viral prepared product of hjolomorphismization of deactivation to prepare dosage form in bulk, it is that the method that adopts embodiment 1 to describe obtains.After the inactivation of virus step, the rabies virus prepared product of this purification and deactivation goes up the excipient that is called 488TM by use at 10KDa film (Omega medium screen PALL) and carries out diafiltration, and this excipient is culture medium 488 preparations that consist of following (seeing table) with it:

Component	Volume/amount
		The mixture of essential amino acids and non essential amino acid	200ml
Sorbitol (Roquette)	50g
		Arginine monohydrochloride (Jerafrance)	10g
EDTA(Prolabo)	0.37g
		Sodium glutamate (SAFC)	4g
Urea	10g
		Tris Na H ₂PO4.2H ₂O(SAFC)	2.42g
Demineralized water QSP	1000ml
		6N hydrochloric acid (Sanofi) QSP	pH=8.0

Mixes with the non essential amino acid solution of 2.5 liters of embodiment, 1 preparation by essential amino acids solution 2.5 liters of embodiment 1 preparation, and by with 30% sodium hydroxide solution with its pH regulator extremely about 7.2 prepare this must with the mixture of non essential amino acid.

488TM is composed as follows for this excipient:

Component	Volume/amount
		Culture medium 488	400ml
Maltose (Hayashibara)	50g
		Tris Na H ₂PO ₄.2H ₂O(SAFC)	1.45g
Demineralized water QSP	1000ml
		6N hydrochloric acid (sanofi) QSP	pH=8.0

The vaccine production thing that obtains is the dosage form in bulk in excipient 488TM, and it is about 12UI/ml based on the virus titer that adopts ELISA to measure glycoprotein G concentration.This dosage form in bulk is preserved with frozen form under-70 ℃, when preparation freeze dried vaccine compositions.

Just before step of freeze drying, a part of dosage form in bulk is carried out freezing and dilution, so that by using following excipient that its last glycoprotein G titre is transferred to about 8UI/ml:

-or excipient F ' 04, it is 1 volume excipient 488TM that its composition contains per 3 volume 50mM phosphate buffers, with the concentration adjustment of maltose to 50g/l, pH=8.0;

-Perhaps excipient F ' 05, and it is identical with excipient F ' 04, but toward wherein adding poloxamer 188 with the 0.01g/l final concentration.

Then, according to the 0.4ml/ bottle two kinds of vaccine combinations are assigned in the lyophilizing bottle, carry out common lyophilization cycle again, this cycle is included in temperature＜-40 ℃ following freezing stage, follow two continuous drying stages, first dryer section is being carried out under-15 ℃ of temperature and the about 80 microbar pressure approximately, and second dryer section is being carried out under+40 ℃ of temperature and the about 80 microbar pressure approximately.

Each lyophilizing is bottled dosage vaccine combination to be tested.The difference of resulting these freeze dried vaccine compositionss only is, has been diluted in according to this dosage form in bulk to have or do not exist this surfactant among excipient F ' 04 or the F ' 05.

Being listed in the excipient that contains in the freeze-dried formulation vaccine combination of a dosage by excipient F ' 05 preparation below forms

Component	Amount (mg/ dosage)
		The mixture of essential and non essential amino acid	0.12
Poloxamer 188	0.004
		Maltose	20.00
Arginine monohydrochloride	0.41
		Sorbitol	2.05
The EDTA disodium	0.01
		Urea	0.41
Sodium glutamate	0.16
		Na ₂HPO ₄.2H ₂O	2.53
NaH ₂PO ₄.2H ₂O	0.12
		Tris	0.24
HCl	QSP
		NaOH	QSP

2.3.2: the effectiveness of freeze dried vaccine compositions

Use two kinds of different stabilising carriers to prepare two kinds of freeze dried vaccine compositionss, adopt NIH official testing method to detect their effectiveness then.Analyzed the effectiveness of two kinds of freeze dried vaccine compositionss, perhaps carried out at once after the lyophilizing (J0), perhaps carried out after one week of preservation down, perhaps after preserving 1 month under 37 ℃, carried out at 45 ℃.Adopt unique official recognition's active testing, the NIH test, it can be determined at the UI number that contains in each test vaccine composition dosage.If UI is number 〉=2.5UI, this test vaccine dosage is effective.NIH test is all carried out two times for each test condition at every turn, optionally takes out two lyophilizing bottles by in preparation under similarity condition and the bottle preserved batch, and these extracts is carried out NIH test and carry out.The scheme that adopts is corresponding to the 0216th piece of monographic scheme of European Pharmacopoeia.This test is the inspectability test of carrying out in mice, its protection that provides based on the test vaccine by this dosage and comparison with the protection that provides with reference to antirabic vaccine of known quantity.This is fractionated with iu (UI) with reference to antirabic vaccine.Iu (UI) is the activity that contains in true quantitative international standard.OMS has set up the equivalent (UI) of international standard.The control parameter that confirms the test of this effectiveness obtain fine satisfy after, determine the UI number that in this test vaccine, contains by the extract of each test with reference to the 50% protective agent value (with the UI classification) that prepared product obtains.

Under the results are shown in that obtains in the Table V.

Table V: the freeze dried vaccine compositions is renderd a service with use

The variation of freeze-dried excipient compositions and preservation condition

The freeze-dried composition holding time (my god) and temperature	J0/+4℃	J7/ +45℃	J30/ +37℃
				F’04	0.8* (0.4; 1.8)**	0.9 (0.4; 2.1)	NT
F’05	6.1 (1.6; 41.3)	2.8 (0.9; 7.9)	3.5 (1.4; 8.7)

*: the meansigma methods of representing with UI that obtains (all test mices groups have 16 mices)

*: the resulting extreme value that expression is represented with UI

NT: do not test out.

The result who discusses in these results and the epimere (2.2) is consistent, and shows that poloxamer 188 should be included in and be used to preserve among the vehicle composition of these freeze dried vaccine compositionss.In fact, these freeze dried vaccine compositionss that only contain excipient F ' 05 (it contains poloxamer 188) contain the vaccine of effective dose, because its average UI value is higher than 2.5UI.These vaccine combinations still are stable, because their effectiveness as time passes and does not reduce under the preservation condition of these tests.

Briefly, these results that list among the embodiment 2 show, these vaccine combinations that contain this rabies virus can obtain fine preservation with stabilising carriers, this stabilising carriers form the mixture that contains phosphate buffer, essential and non essential amino acid, sucrose (as maltose), polyhydric alcohol (as Sorbitol), EDTA, urea and with the nonionic surfactant of its combination, as poloxamer 188.In addition, advantageously, the Stabilization of poloxamer 188 is brought into play under low-down concentration, and this concentration was low for activating this immune system or being used as immune response modifier.

Embodiment 3: the stability study of vaccine combination that contains the rabies virus of purification and deactivation

Embodiment

1 and 2 has shown the importance of the composition that makes excipient and poloxamer 188 combinations, this excipient contains mixture, maltose, Sorbitol, EDTA, the urea of essential and non essential amino acid in the buffer based on phosphate and/or Tris, the purpose of introducing research here be the wide storage temperature range of card all too (+37 ℃ ,+25 ℃ ,+5 ℃ ,-70 ℃) in in the long time section, this combination makes the rabies virus vaccine combination of different dosage form (liquid, freezing or lyophilizing) stable effectively.These researchs are carried out multiple rabies virus vaccine combination, these rabies virus vaccine combinations are preserved down in temperature≤-35 ℃ with freezing dosage form in bulk, with final liquid bulk products dosage form+5 ℃ down or with the unit freeze-dried formulation+5 ℃ ,+25 ℃ or+37 ℃ preserve down.

3.1 the stability study of freezing dosage form antirabic vaccine in bulk batch

Prepared the rabies virus that is used to prepare dosage form in bulk batch according to as described in Example 1 method.After the inactivation of virus step, the viral prepared product of purification and deactivation carries out diafiltration by use 50mM phosphate buffer as diafiltration buffer on film 10KDa.Secondly, trapped substance and 1 volume stability mixed with excipients with 1 volume, the composition of this stabilising carriers is the composition of the buffer 489PM of embodiment 1 description, and (except each component concentrations is a twice) is toward the poloxamer 188 that wherein adds concentration 0.02g/l, pH ≈ 8.0.Obtaining having the dosage form in bulk of 50 μ g/ml total protein concentrations, wherein is rabies virus albumen more than 70%, and it contains the following residual DNA of 100pg/ml.At-35 ℃ with preserve down the back at-70 ℃ and measured glycoprotein G titre by per 3 months and studied the stability of this dosage form in bulk as time passes.Under the results are shown in that obtains in the Table VI.

Table VI: the stability of the dosage form in bulk of preserving down-35 ℃ or-70 ℃ batch

Storage temperature	T0	T0+3 month	T0+6 month	T0+9 month	T0+12 month
						-35℃	20.4*	20.84	24.4	24.3	22.4
-70℃	21.3	NT	22.4	22.8	21.8

*: titre, represent with UI/ml

NT: do not test out.

Do not observe the glycoprotein G degraded of the major antigen that is this rabies virus after storing 12 months under-35 ℃ or-70 ℃, this has confirmed the good stability of freezing dosage form vaccine combination in bulk.In addition, after-35 ℃ or-70 ℃ are preserved 12 months, adopt DLS to the analysis showed that of virion distribution of sizes, these curves kept with intermediate value 180nm be the center gaussian shape (with the class of a curve of Fig. 1 and 2 like).Do not exist and show these curve divisions that have viral aggregation to exist.These results show that also the rabies virus population does not change as time passes.Do not observe described virion degraded and gathering as time passes.

3.2: the stability study of liquid finished dosage forms in bulk (PFV) antirabic vaccine batch.

Use 3 dosage forms in bulk batch in buffer 489PM (composition of describing among the embodiment 1), to prepare the vaccine batch of 3 liquid PFV dosage forms after the dilution, in this buffer 489PM, added poloxamer 188 (0.01g/l final concentration, pH ≈ 8.0).They contain the following residual DNA of 100pg/ml (adopting quantitative PCR to measure).Total protein concentration is that about 15 μ g/ml and 70% above total protein are virus proteins behind the electrophoresis photodensitometry on the polyacrylamide gel.By adopt ELISA in 3 months to the PFV batch of content of measuring glycoprotein G termly+5 ℃ of preservations, with in 30 day time to the PFV batch of content of measuring glycoprotein G termly+25 ℃ of preservations, studied PFV batch of stability+5 ℃ and+25 ℃ of preservations.The result who obtains represents with UI/ml, and collects in down Table VII and VIII.

Table VII: in 3 PFV batch stability of+5 ℃ of preservations

Lot number	T0	T0+1 month	T0+2 month	T0+3 month
					N°1	10.5	12.0	8.6	10.8
N°2	11.1	10.7	9.3	11.9
					N°3	13.9	11.1	11.6	11.2

Table VIII: in 3 PFV batch stability of+25 ℃ of preservations

Lot number	T0	T0+15 day	T0+30 day
				N°1	12.0	10.9	10.9
N°2	10.7	11.4	10.1
				N°3	11.1	12.0	11.5

These results show, in the time period of being studied liquid dosage form PFV batch+5 ℃ and+25 ℃ storage life between, do not have glycoprotein G degraded.This confirmation, these antirabic vaccines can be preserved 3 months at low temperature (+5 ℃) with liquid dosage form at least, room temperature (+25 ℃) at least 30 days, and observed this virus degraded.

3.3: the stability study of freeze-dried formulation antirabic vaccine batch

2 freeze-dried formulation antirabic vaccines batch (LL1 and LL2) are with the PFV batch of preparation of embodiment 3.2, according to the 0.3ml/ bottle, they are distributed in the lyophilizing bottle made from glass, carry out lyophilization cycle then, in its process, under≤-40 ℃ temperature, carry out freezing, first dryer section under about-15 ℃ of temperature and about 80 microbar pressure up to ice distillation fully, and second dryer section under+40 ℃ of temperature of pact and about 80 microbar pressure up to residual moisture content≤3% in these lyophilized products.Virus titer loss＜5% when step of freeze drying.

The excipient that in being the vaccine dose of freeze-dried formulation, contains composed as follows:

Component	Amount is represented with mg/ dosage
		The mixture of essential and non essential amino acid	0.37
Poloxamer 188	0.003
		Maltose	15.00
Arginine monohydrochloride	1.20
		Sorbitol	6.00
The EDTA disodium	0.04
		Urea	1.2
Sodium glutamate	0.15
		Na ₂HPO ₄.2H ₂O	2.53
NaH ₂PO ₄.2H ₂O	0.12
		HCl	QSP
NaOH	QSP

These lyophilizing dosage units that obtain have the uniform outer appearance of white.The pH that obtains the rabies virus suspension after dissolving these lyophilized products again with 0.5ml 0.4% sodium chloride solution is 7.8 ± 0.5.Studied stability in 3 storage temperature 35-39 ℃, 23-27 ℃ and 2-8 ℃ two lyophilizing as time passes batch, by verifying that lyophilized products is in the outward appearance of reconstruct front and back, by measuring residual moisture, by controlling its pH, virus titer, and undertaken by the effectiveness that detects lyophilized preparation in the NIH test.Be diluted in 0.9% sodium chloride solution that contains the 2g/l human albumin first time of dilution series (it carries out in order to detect described lyophilized preparation be dissolved in sodium chloride in the NIH test after) carries out.

Adopt the ELISA method based on glycoprotein G assay these virus titers (representing) with UI/ml.

About the test result of lyophilized products outward appearance and residual moisture and pH measurement result be with specificity duplicate (promptly this lyophilized products be even white appearance, colourless transparent solution after the reconstruct in the hypo-osmoticity physiological serum, and residual moisture content＜3%), how also all like this storage temperature of being tested and time is.PH still is in 7.8 ± 0.5 scopes in during stability study whole.

The result of the effectiveness test of carrying out about these virus titers with to described unit dose (NIH) collects among the following table IX-XI.

Table I X: in the stability of+5 ℃ of two antirabic vaccines preserving with freeze-dried formulation batch

Table X: in the stability of+25 ℃ of two antirabic vaccines preserving with freeze-dried formulation batch

Table X I: in the stability of+37 ℃ of two antirabic vaccines preserving with freeze-dried formulation batch

M: the expression moon

ND: the expression undetermined goes out

*: the value of representing with UI/ml

*: with the meansigma methods that the UI numerical table shows, its completion the observed extreme value of in bracket, listing.

All these results confirm, these antirabic vaccines of preserving with freeze-dried formulation are preserved highly stable, and do not have loss of effectiveness as time passes under these probe temperature conditions.These glycoproteins G titre and NIH test value (apparently higher than 2.5UI) are preserved stable as time passes.

Conclusion: a plurality of antirabic vaccines batch all stability tests that carry out of preserving with freezing dosage form, liquid dosage form or freeze-dried formulation are shown it is stable that these batches keep as time passes under the condition of these probe temperatures.This confirmation, its composition contain buffer, must and the excipient of mixture, maltose, Sorbitol, EDTA, urea and the poloxamer 188 of non essential amino acid make the antirabic vaccine composition stable effectively, how and all like this under the storage temperature of wide region its existing way (freezing, liquid or lyophilizing).

Claims

1. vaccine combination, it contains:

A) the totivirus prepared product of deactivation, and

B) stabilising carriers, it comprises:

I. buffer solution,

The mixture of ii. essential and non essential amino acid,

Iii. disaccharide,

Iv. polyhydric alcohol,

V. chelating agen,

Vi. urea or urea derivative, and

Vii. nonionic surfactant.

2. according to the compositions of claim 1, the totivirus of wherein said deactivation is a rabies virus.

3. according to the compositions of claim 1 or 2, it is characterized in that described compositions is also without any serum albumin.

4. according to each compositions among the claim 1-3, it is characterized in that described compositions is also without any the foreign protein of animal origin, preferably without any the outer product-derived of animal origin.

5. according to each compositions among the claim 1-4, it is characterized in that virus protein accounts at least 70% of the total protein that exists in this vaccine combination.

6. according to each compositions among the claim 1-5, it is characterized in that total protein concentration is≤100 μ g/ml, preferably≤80 μ g/ml.

7. according to each compositions among the claim 1-6, it is characterized in that the amount of the total protein that is contained is≤100 μ g in the vaccine combination of an effective dose.

8. according to each compositions among the claim 1-7, it is characterized in that the amount of the total protein that is contained is 1-50 μ g in the vaccine combination of an effective dose.

9. according to each compositions among the claim 1-8, it is characterized in that described stabilising carriers is without any albumen, perhaps without any albumen and any peptide, perhaps preferably without any albumen, any peptide and any oligopeptide.

10. according to each compositions among the claim 1-9, it is characterized in that, described must and the mixture of non essential amino acid comprise arginine or arginine salt and glutamic acid or glutamate, Glu at least.

11. according to each compositions among the claim 1-10, it is characterized in that, in the vaccine combination of an effective dose, contained must and the amount of non essential amino acid be 0.5-2.5mg.

12., it is characterized in that described disaccharide is a maltose according to each compositions among the claim 1-11.

13., it is characterized in that described polyhydric alcohol is a Sorbitol according to each compositions among the claim 1-12.

14., it is characterized in that the disaccharide that is contained and the total amount of polyhydric alcohol are 10-50mg in the vaccine combination of an effective dose according to each compositions among the claim 1-13.

15., it is characterized in that described intercalating agent is EDTA or edta salt according to each compositions among the claim 1-14.

16., it is characterized in that the amount of the intercalating agent that is contained is 0.01-0.1mg in the vaccine combination of an effective dose according to each compositions among the claim 1-15.

17., it is characterized in that the urea that is contained or the amount of urea derivative are 0.3-1.5mg in the vaccine combination of an effective dose according to each compositions among the claim 1-16.

18., it is characterized in that described nonionic surfactant is a poloxamer according to each compositions among the claim 1-17.

19., it is characterized in that described nonionic surfactant is a poloxamer 188 according to each compositions among the claim 1-18.

20., it is characterized in that the amount of the nonionic surfactant that is contained is 0.001-0.5mg in the vaccine combination of an effective dose according to each compositions among the claim 1-19.

21., it is characterized in that the pH of this vaccine combination is 7.0-10.0, preferably 7.0-9.0 according to each compositions among the claim 1-20.

22., it is characterized in that described buffer solution is phosphate buffer, Tris buffer, HEPES buffer or their mixture according to each compositions among the claim 1-21.

23., it is characterized in that described buffer solution is the phosphate buffer of 10-100mM for its molar concentration according to each compositions among the claim 1-22.

24. prepare the method for vaccine combination, described vaccine combination contains the totivirus prepared product of purification and deactivation, wherein:

A) produced the totivirus prepared product by results by the supernatant of the cell culture of described viral infection,

B) described totivirus prepared product is carried out purification and deactivation, perhaps alternatively with described totivirus prepared product deactivation purification then, and

C) the totivirus prepared product with described purification and deactivation is diluted in the stabilising carriers, and the composition of described stabilising carriers comprises:

I. buffer solution,

The mixture of ii. essential and non essential amino acid,

Iii. disaccharide,

Iv. polyhydric alcohol,

V. chelating agen,

Vi. urea or urea derivative, and

Vii. nonionic surfactant.

25. the method according to claim 24 is characterized in that, implements this method under the situation of the outer product-derived of not introducing animal origin.

26. the method according to claim 24 or 25 is characterized in that, described virus is rabies virus.

27., wherein after the totivirus prepared product of described purification of dilution and deactivation, the vaccine combination that so obtains is distributed in the packing device, and randomly with this vaccine combination lyophilizing according to each method among the claim 24-26.

28. the purification that exists with freeze-dried formulation that vaccine combination, its method that contains with good grounds claim 27 obtain and the totivirus prepared product of deactivation.

29. be used for the stabilising carriers of the whole virus vaccine of deactivation, its composition comprises:

I. buffer solution,

The mixture of ii. essential and non essential amino acid,

Iii. disaccharide,

Iv. polyhydric alcohol,

V. chelating agen,

Vi. urea or urea derivative, and

Vii. nonionic surfactant.

30. the stabilising carriers according to claim 29 is characterized in that, described stabilising carriers is also without any albumen, perhaps without any albumen and any peptide, perhaps preferably without any albumen, any peptide and any oligopeptide.

31. the stabilising carriers according to claim 30 is characterized in that, described stabilising carriers is also without any the product of animal origin.